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Tande m Mass Spectrometry - Applications and Principles

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HPLC-MS/MS

of Highly Polar Compounds

Luigi Silvestro1, Isabela Tarcomnicu2 and Simona Rizea Savu1

13S-Pharmacological Consultation & Research GmbH, Harpstedt,

2Pharma Serv Int’l SRL, Bucharest,

1Germany

2Romania

1. Introduction

Complicate high-performance liquid chromatographic (HPLC) interfaces like the moving

belt, direct liquid interface, thermospray (Arpino, 1985) fast atom bombardment (FAB)

(Caprioli et al., 1986; Garcia & Barceló, 1993; Dell et al., 1988; Larry et al., 1989) characterized

the early days of HPLC used in tandem with mass-spectrometry (MS); all of them, apart

FAB, were more effective with moderately polar compounds (Strege et al., 1999). After years

of difficulties and struggles the introduction of atmospheric pressure ionization by

electrospray (ESI) or chemical ionization marked a clear breakthrough: an effective, robust

and user friendly HPLC interface was finally available.

As a matter of fact, considering the physicochemical principles of the source, the polar

compounds were the first compounds successfully ionized (Griffits, 2008).

Despite the huge potential of ESI for polar and highly polar compounds the applications for

the latter proved to be critical due to the fact that effective HPLC separations often require

non volatile buffers and/or high water content that are not suitable in MS detection.

Along the years, the development of HPLC columns, specific derivatization methods, new

chromatographic techniques and sample preparation approaches have increased the

number of applications with highly polar compounds. Three classes of not so widely

studied molecules have been selected in this study in order to illustrate the application of

HPLC-MS/MS to highly polar compounds; updates as well a review of past data will be

presented.

The groups of compounds taken in consideration are: glycosaminoglycans, bisphosphonates

and amynoglycoside antibiotics; the first-ones are both endogenous and exogenous while

the last 2 classes are of typical pharmaceutical interest.

Obviously other more well–known molecules like peptides and polynucleotides are also

included in this class of highly polar compounds but HPLC-MS/MS methods in these

particular fields are widely described and probably represent the most common areas of

application. It is also important to keep in mind that this chapter doesn’t intend to be an

exhaustive presentation of the published analytical methods for heparins, bisphosphonates

or aminoglycosides, but these compounds are used as models to emphasize the potential of

HPLC-MS/MS in the analysis of highly polar molecules.

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2. Glycosaminoglycans

2.1 Chemical structure and main features of glycosaminoglycans

Glycosaminoglycans (GAGs) are a family of highly sulphated, complex, polydispersed

linear polysaccharides that display a variety of important biological roles. They contain

amino-sugars with alternating disaccharide units, which comprise an acidic monosaccharide

such as glucuronic or iduronic acid and a basic monosaccharide such as N-

acetylglucosamine or N-acetyl-galactosamine; both monosaccharides can be sulphated. In

natural biological systems GAGs are mainly linked to proteins to form proteoglycans, with

great interest for medical science being structural proteins or receptor as well adhesion

molecules (just to mention a few examples) and can be categorized, based on the GAGs

structure, into three main structural groups: heparin/heparan sulphate group,

dermatan/chondroitin sulphate group and hyaluronan (Mao et al., 2002; Tiayu Peng, 2002).

Heparin and heparan sulphate occur as proteoglycans in which the glycosaminoglycan chains

have different lengths and are composed of disaccharide units consisting of L-iduronic or D-

glucuronic acids (1-4) linked to glucosamine units that can be N-acetylated or N-sulphated

(Figure 1).

Heparin and heparan sulphate have closely related structures and consist of similar

disaccharide composition except that heparin has a greater content of iduronic acid and is

more highly sulphated per polysaccharide chain than is heparan sulphate. The differences

between the chemical structure of the sequences of heparin and heparan sulphate explain

the biological function related to them. As an example, the specific interaction of heparin

with the protein antithrombin III, determining its anticoagulant activity, is mediated by a

characteristic pentasaccharide that is not normally present in heparan sulphate and it

accounts for just a few percent of the heparin mass (Saad & Leary, 2003).

Fig. 1. Main sequence structure of heparin/heparan sulphate.

The dermatan/chondroitin sulphate are the main components of connective tissues and the

chemical structure comprises alternating uronic acid and N-acetyl-galactosamine residues;

depending on their composition 3 main types of chondroitin sulphates have been identified

(Gunay & Linhardt, 1999). The structures of these types of chondroitin sulphate are

presented in Figure 2.

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H2COR2

COOH

R1O

NHCHCH3

Chondroitin Sulphate Type A: R=H, R1=SO3H, R2=H, R3=COOH

Chondroitin Sulphate Type B: R=COOH, R1= SO3H, R2=H, R3=H

Chondroitin Sulphate Type C: R=H, R1=H, R2=SO3H, R3=COOH

Fig. 2. Structure of chondroitin sulphate.

Chondroitin sulphate A and chondroitin sulphate C contain D-glucuronic acid, N-

acetylgalactosamine and sulphate residues in equimolar quantities but differ in the position

of the sulphate ester group (Saito et al., 1968). Dermatan sulphate formerly called

chondroitin sulphate B is similar to chondroitin sulphate A but instead of D-glucuronic acid

it consists mainly of L-iduronic acid.

Hyaluronic acid has alternating repeating units of the structure, (1→3) -N-acetyl-D-

glucosamine (1→4) -D-glucuronic acid, but tipically it is not sulphated and the molecules

are larger than the other glycosaminoglycans, often several million Da, with special

rheologic characteristic (Mao et al., 2002).

Fig. 3. Structure of the repeating units of Hyaluronic acid.

2.2 Special problems of these molecules and potential approach to solve them

GAGs are extremely difficult to analyze because of their polydispersity and

microheterogeneity of chemical structure combined with high polarity. The first two aspects

(polydispersity and microheterogeneity) push to get an optimal separation of all species

presented in a sample in order to may accurately characterize each component. It is in fact

important to consider that these molecules are closely related polymers with common

disaccharides/oligosaccharides and if not well separated it is also impossible to get detailed

structure information.

In the meantime the high polarity of GAGs make them difficult compounds for optimal

chromatographic separation and an adequate separation of all components present in a

GAGs sample is virtually impossible.

In such conditions two approaches are feasible:

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1. Application of analytical techniques able to gather structure data also of unfractionated

or partially fractionated complex mixtures

2. Structure specific degradation of the GAGs in order to reduce such complex samples

with almost infinite variant of similar polymers to more discrete mixtures of oligomers

corresponding to their “building blocks” that can be properly analyzed and quantified

obtaining quite detailed information of the polymers themselves intended as a mean

structure or a fingerprint.

Regarding the first approach, only NMR permits to get in-depth chemical structure

information of almost unfractionated materials but the instrumental sensitivity is a major

drawback when studying limited biological samples and in any case the information

obtained are practically a mean of the individual structures of the components present in the

sample. Despite the fact that hyphenation of NMR and HPLC (as separation method) is

feasible, due to sensitivity problems its application on such complex samples is more

theoretical then practical so far.

Before to close this parenthesis on NMR, outside the scope of this chapter, it is evident that

NMR spectroscopy is also a very effective technique for the characterization of the

oligosaccharides (Yung, 2011). It gives valuable information on monosaccharide

composition, glycosidic linkage, uronic acid type and sulfation patterns (Bo et al., 2011), but

it requires always relatively large amount of material and is not suitable for the analysis of

very small samples, like most biological samples. By coupling capillary isotachophoresis

with on-line microcoil NMR detection (CITP-NMR) the sensitivity for the characterization

of heparin – derived oligosaccharides has been improved (Korir & Larive, 2007).

Considering the limitations related to this first analytical approach, based on intact

molecules, the focus in the next paragraphs will be on presenting methods to analyze GAGs

following depolimerization; clearly that in rare cases of small oligosaccharide (i.e. from

synthesis) or highly purified fractions such step can be avoided.

2.3 Overview on methods of GAGs depolimerization

As previously pointed out most often the structure characterization of GAGs involves

degradation steps to obtain disaccharides and/or a range of oligosaccharide fragments

allowing an efficient disaccharide/oligosaccharide mapping (Bo et al., 2011). These

structural analyses of GAGs may involve complete or partial chemical degradation, as well

as enzymatic degradation permitting a quite selective cut of the polysaccharide chains

(Ruiz-Calero et al., 2003; Gatti et al., 2010). Highly specific enzymes have been isolated and

they are now commercially available making their application viable (Johnson, 1982; Jandik

et al., 1996; Cohen & Linhardt, 1990). In figure 4 the main used enzymes are shown.

An important aspect of these enzymes is the high specificity for the substrate, fact that if it is

welcomed in biochemical studies can be critical sometimes when analyzing semisynthetic

derivatives.

Chemical degradation is a very interesting alternative to enzymatic degradation giving also

quite specific structure information. The degradation with nitrous acid and the periodic

oxidation followed by Smith degradation are two classical tools for GAGs analysis.

In several cases depolimerization methods are followed by derivatization procedures

generally to enhance the analytical detection when using conventional HPLC or CE

detectors.

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(A) Heparin and heparan sulphate

(B) Chondroitin sulphate (C) Hyaluronic acid

Fig. 4. The enzymatic cleavage of GAGs; A) heparin, heparan sulphate and the specific

enzymes: Heparinase I, Heparinase II and Heparinase III; B) Chondroitin sulphate and the

specific enzymes: Chondroitinase ABC for Chondroitin sulphate A, B and C, Chondroitinase

AC for Chondroitin sulphate A and C and Chondroitinase C for Chondroitin sulphate A

and C; C) Hyaluronic acid and the specific enzyme: Hyaluronidase

2.4 Overview on HPLC analysis of GAGs

For the analysis of intact GAGs, a variety of chromatographic techniques have been

developed mainly to separate different group of GAGS and/or to characterize the molecular

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weight distribution. Concerning this last aspect gel permeation chromatography (both as

HPLC or low pressure chromatography) is the preferred approach.

Ion exchange chromatography and reversed phase ion-pair chromatography are the most

employed techniques to separate the main classes of intact GAGs, which present differences

in polarity, both as low pressure chromatography (Patel et al., 2009; Jin et al., 2009) or high

performance liquid chromatography (Shao et al., 2004; Trehy et al., 2009). More recently also

capillary electrophoresis (CE) (Malsch & Harenberg, 1996; Malsch et al., 1996) have been

used successfully. The main interest is, however, on methods for the separation of

depolymerisation fragments of GAG, obtained by different digestion methods (chemical or

enzymatic), by HPLC (Du & Eddington, 2002; Plaas et al., 1996; Toyoda et al., 2000) or CE

(Malavaki et al., 2008; Ruiz-Calero, et al., 2003) followed by detection with conventional

spectral system (UV or fluorescence in case of adequate derivatization), as in the previous

papers, but in the last years often by mass spectrometry (Oguma et al., 2007; Yang et al.,

2011; Barosso et al., 2005; Silvestro et al., 1992; Da Col et al., 1993; Hemstrom & Irgum,

2006; Silvestro et al., 1996).

The peculiar aspects of separation in connection with MS detection will be detailed in the

following paragraph.

2.5 Overview on mass-spectrometric analysis of GAGs

Methodologies using fast atom bombardment (FAB) were first employed; however a direct

coupling to HPLC separation was not achieved, as in the case of peptides, due to limitations

in mobile phase composition (Dell et al., 1988; Mallis et al., 1989). The introduction of soft

ionization methods (ESI), permitting an easy interfacing to HPLC has first provided an on-

line separation and structure elucidation of complex oligosaccharide mixtures (Henriksen et

al., 2006; Barosso et al., 2005; Thanawiroon et al., 2004). As a complement, matrix-assisted

laser desorption ionization (MALDI) has proved to be a valuable tool for the analysis of

protein/peptide-heparin/HS oligosaccharide complexes (Ori et al., 2009; Venkataram et al.,

1999).

The first important point to consider is the kind of oligosaccharides and disaccharides to be

analyzed in particular if they are sulphated or not sulphated.

2.5.1 Sulphated oligosaccharides

As discussed previously (Chapter 2.4), sulphated oligosaccharides and disaccharides are

separated, when using conventional detectors (UV, fluorescence, electrochemical),

employing strong ion exchange columns with gradients of molarity of salts from strong

acids (sulphates, phosphates, chloride etc.). It is evident that such mobile phases, being non-

volatile, cannot be used in case of MS interfacing and alternative solutions have been

developed.

An option is the application of weak anion exchange columns permitting the elution of

sulphated derivatives also with gradients of ammonium acetate, compatible with an ESI

source; nonetheless it cannot be excluded that highly sulphated large oligomers are not

eluted from this kind of columns.

Another possibility, probably the most widely used, is the separation on a reversed phase

column in presence of an ion-pairing reagent. In an early paper by our group (Da Col et al.,

1993) tetrapropylammonium (TPA) was selected as ion pair reagent for this kind of

separation; smaller quaternary ammonium derivatives (tetramethylammonium and

tetraethylammonium) didn’t improve at all the chromatographic separation while larger

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derivative (tetrabutylammonium and higher) showed critical problems of volatility in the

ionization source without offering other advantages. It is important to note that the choice of

the stationary phase can be quite critical: we observed in fact that in case of reversed phase

columns with longer aliphatic chain (C8 or longer) the sulphated oligosaccharides couldn’t

be eluted being probably retained by an HILIC-like retention mechanism. Best conditions

were obtained on C4 – C6 columns with gradients of acetonitrile water (from 100% to 50%

water) in presence of 3.3 mM tetrapropylammonium at pH 4.5 with formic acid. To prepare

the mobile phase most common tetrapropylammonium salts like the sulphate (common in

conventional HPLC) were avoided due to volatility problems and preparations were made

starting from tetrapropylammonium hydroxide. An example of TIC chromatograms

recorded on a digest of heparin is presented in Figure 5.

More recently, several groups have successfully introduced the use of tertiary aliphatic

amines (tributylammonium acetate in acetonitrile as ion pair reagents) claiming that a better

volatility can be obtained (Thanawiroon et al., 2004). It must be observed that short chain

amines like triethylamine, widely used in HPLC, are not able to form ion-pair with these

sulphated oligosaccharides.

Fig. 5. Total ion chromatogram obtained in TOF full scan after injecting a sample of heparin

digested with heparinase II. Column: Kromasil C4 (100x2.1mm, 3μm); mobile phase: (A)

TPA 3.3 mM in water, pH 4 and (B) TPA 3.3 mM in acetonitrile/water (90/10, v/v), pH 4.

Mass spectrometer: QTOF Qstar XL, operated in negative ESI mode.

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The addition of an ion pair reagent to sulphated oligosaccharides brings to the formation of

complexes stable enough to be ionized as such. Complex pseudomolecular ions with one or

more molecules of the ion-pair reagent, sometimes multiple charged, are predominant in

spectra of chromatographic separations of such di- and oligo-saccharides; 2 examples of

similar spectra are presented in Figure 6 and Figure 7.

Fig. 6. The molecular ion of the adduct formed by the disaccharide Heparin III H and TPA

[M+TPA-H]- = 601.2439. Spectrum recorded by direct infusion with the syringe of a solution

diluted at 1 μg/mL. Mass spectrometer: QTOF Qstar XL, operated in negative ESI mode.

It is evident that the use of high resolution instruments is a valuable tool to clarify the

charge state of these ions while to clarify the stoichiometry of ion-pair

reagent/oligosaccharide ratio the utilization of a labelled ion-pairing reagent is very

interesting (Silvestro et al., 1996).

In an effort to obtain more easily interpretable spectra an attractive approach is also the use

model MMPC of the ion suppressor (i.e. Dionex…) to remove the tetrapropylammonium

from the chromatographic mobile phase. The experiments carried out have really showed an

efficient removal of tetrapropylammonium (an example is shown in Figure 8). Anyway, the

oligosaccharides as free acidic compounds are much more prone to non-sequence related in

source fragmentation during ionization (mainly desulphation), making sometimes very

difficult to understand the oligosaccharide real structure.

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Fig. 7. Extracted spectrum obtained after injecting a sample of heparin digested with

heparinase II. Column: Kromasil C4 (100x2.1mm, 3μm); mobile phase: (A) TPA 3.3 mM in

water, pH 4 and (B) TPA 3.3 mM in acetonitrile/water (90/10, v/v), pH 4. Mass

spectrometer: QTOF Qstar XL, operated in negative ESI mode. M: trisulphated disaccharide

obtained from heparin by enzymatic digestion.

The MS/MS properties of these di- and oligo-saccharides are also noteworthy: in case of

parent ions (single or multiple charged) the desulphation fragments are generally the most

abundant being easily formed and generally very few weak signals coming from

fragmentation of the sugar back bone can be observed. The situation is similar in case of

TPA complexes with the tendency to an even less favourable fragmentation.

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Fig. 8. The molecular ion of the oligomer Heparin III H after ion suppression; [M-H]- =

416.1532 can be easily seen. Spectrum recorded by direct infusion with the syringe of a

solution diluted at 1 μg/mL. Mass spectrometer: QTOF Qstar XL, operated in negative ESI

mode.

2.5.2 Non sulphated oligosaccharides

The analytical separations of these molecules are less challenging than those of the more

charged sulphated products. In this case the use of ion exchange columns eluted with

gradients of volatile buffers (i.e. ammonium acetate) at high ionic strength is effective and

permits a good separation of oligomers with different chain length. The application of ion-

pair chromatography is practically not needed and in general the formation of ion-pair

complexes having only carboxylic groups is not effective.

Deprotonated molecular ions, sometimes multiple charged, are typically observed, as shown

in Figure 9.

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Fig. 9. Mass spectrum obtained in TOF full scan mode after injecting a sample of a

hyaluronic acid digested with hyaluronidase. Column: Supelcosil LC-SAX 1 (250x4.6mm,

5μm); mobile phase: (A) water/methanol (1/1, v/v) (B) ammonium acetate 0.5 M in

water/methanol (1/1, v/v). Mass spectrometer: QTOF Qstar XL, operated in negative ESI

mode. The main ions in the spectrum are multiple charged; they all correspond to

dissacahride oligomers resulted from hyaluronic acid degradation. A detail can be seen in

the spectra above for [M-2H]2-=1136.8, a doubly charged ion corresponding to a

dodecasaccharide.

Daughter spectra can be obtained as it is shown in Figure 10. However, it can be seen, these

kinds of molecules are hard to break and only scarce structural information can be gathered.

3. Bisphosphonates analysis

3.1 Chemical structure and main features of bisphosphonates

Bisphosphonates, polar structures with two phosphonate groups covalently bonded to

carbon (called P-C-P bridge, Fig. 11) are a class of drugs used in the treatment of

osteoporosis, osteolytic metastasis, Paget’s disease (osteitis deformans), and other

disorders involving bone fragility. They have also non-therapeutic usage, based on their

chelating properties (as water softeners, in agriculture, in paper, detergent and cosmetics

industries).

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Fig. 10. Product ion spectrum of one of the oligomers presented in Figure 9 - [M-2H]2- =

1010. Column: Supelcosil LC-SAX 1 (250x4.6mm, 5μm); mobile phase: (A) water/methanol

(1/1, v/v) (B) ammonium acetate 0.5 M in water/methanol (1/1, v/v). Mass spectrometer:

QTOF Qstar XL, operated in negative ESI mode; collision energy: -30V.

Fig. 11. General structure of bisphosphonates.

The long-chain substituent (R2 in figure) determines the chemical properties and mechanism

of action, while the short-chain substituent (R1) influences mainly the pharmacokinetics of

the bisphophonate drug (Fleish 2004). Obviously, bisphosphonates are extremely

hydrophilic and their structures similar to endogenous phosphorylated compounds. They

can be grouped in two classes: nitrogen (N) – containing (pamidronate, neridronate,

olpadronate, alendronate, ibandronate, risedronate, zoledronate) and non-N-containing

(etidronate, clodronate, tiludronate) bisphosphonates.

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3.2 Special problems of these molecules and potential approach to solve them:

derivatization or not

Analysis of bishposphonates from biological matrices is very challenging, due to the

aforementioned features of these molecules: high polarity and similarity with endogenous

phosphates. Finding appropriate conditions for their extraction from plasma or urine is very

difficult; chromatographic separation raise typical problems of hydrophilic compounds and

last but not least, bisphosphonate molecules often lack chromophores or fluorescent groups

needed for sensitive UV or fluorescence detection in liquid chromatography. As an

approach to facilitate either the isolation from biological samples, or the chromatographic

separation, or the detection, or all of them, derivatization is widely used in the analysis of

bisphosphonates. Mass spectrometric detection was used more recently to develop sensitive

quantification methods, but also most of the times in combination with derivatization

procedures. In gas-chromatography (GC), derivatization is needed to increase the volatility

and thermal stability of the molecules. The N-containing bisphosphonate undergo reaction

of the amino- group, and for all compounds the derivatization of the hydroxyls from the

phosphonate group can give good results.

3.3 HPLC analysis of bisphosphonates

Various analytical procedures for the determination of bisphosphonates from biological

samples or pharmaceutical formulations have been presented so far; review papers were

previously published by Sparidans and den Hartigh (1999), Zacharis and Tzanavaras (2007).

One of the first methods, developed for dichloromethylene bisphposphonates, was

described by Chester et al (1981). The separation is based on ion-exchange chromatography,

without derivatization, using an AG1 X8 resin column and a mobile phase containing

hydrochloric acid and a flame-photometric detector. The coprecipitation with calcium

phosphate was used to extract the target compounds from urine. This method has a limit of

detection of 2µmol/l.

Separation procedures without derivatization are performed on ion-exchange columns like

Waters IC-Pak HR (Tsai et al, 1992) or Dionex OmniPac PAX-100; the mobile phases contain

mineral acids (mostly diluted nitric acid without or with small percentage of organic

modifier) and the detection is realized through flame-photometry (Chester et al, 1981),

conductivity (Tsai et al, 1992) or inductive coupled plasma mass spectrometry (ICP-MS)

(Kovacevic et al, 2004).

Mineral acids are replaced with organic acids (especially formic acid) in researches that use

ion-chromatography in tandem with mass-spectrometry (Ip et al, 1994). An alternative to

ion-exchange chromatography is ion-pairing reversed-phase chromatography. Xie et al

(2006) used ion-pair chromatography with evaporative light-scattering detection to

determine ibandronate in pharmaceutical formulations. The C8 Inertsil column was eluted

in isocratic conditions with a mobile phase consisted of ammonium acetate containing

amylamine/acetonitrile/methanol. Tetrabutyl ammonium hydrogen sulphate (Rao et al,

2005), tetrabutyl-ammonium phosphate (Aluoch et al, 2005), 1-octyltrimethylammonium

phosphate (Vallano et al, 2003) are among the ion-pair reagents employed in separations

without derivatization.

As mentioned above, a pre- or post-column derivatization step is often needed to introduce

in the molecule a chromophore group, in order to increase the sensitivity of the detection by

UV or fluorescence. The pre-column derivatization, if possible directly in biological samples,

is also very important for the isolation of bisphosphonates, introducing in the hydrophilic

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molecule a large non-polar group that facilitates their subsequent extraction in organic

solvents.

Most of the published methods involve therefore a derivatization approach, either of the

amino, or of the hydroxyl groups, or of both of them in some cases.

Fluorescamine was one of the most used derivatization reagents of the amino group (Flesh

et al, 1991, Wong et al, 2004). Aminoalkylphosphonic acids were isolated from urine or

plasma by coprecipitation with calcium phosphate; then the precipitate was dissolved in

Na2EDTA pH10 and reacted with fluorescamine. The reaction products were

chromatographed on an octadecyl stationary phase (Nucleosil C18) and detected by

fluorescence.

9-Fluorenylmethyl-chloroformate was also widely applied in the determination of

alendronate from different matrices like pharmaceutical preparations, plasma or urine. The

derivative was chromatographed on C18 columns, with mobile phases consisting of

acetonitrile, methanol and pyrophosphate buffer, and detection in UV or fluorescence (de

Marco et al, 1989, Ptacek et al, 2002, Yun et al, 2006, Apostolou et al, 2007). As an example,

Ptacek et al (2002) obtained with this derivatization a limit of quantification as low as 5

ng/ml from urine, with reproducible results that could be used for bioavailability studies.

The reaction was conducted at alkaline pH after bisphosphonates isolation from biological

sample by coprecipitation with calcium phosphate and the calcium ions removed on ion-

exchange resins; the chromatography was carried out on a C18 stationary phase, in gradient

conditions, with fluorescence detection.

The derivatization of the amino group with isobutyl chloroformate was proposed by

Sakyiama et al (1996) prior to GC separation. A fused-silica capillary column HP-1 (5m x

0.53 mm i.d., 2.65μm film thickness) was used and flame photometric detection. The same

reaction was later used by our group directly on biological samples (urine); this reduced the

polarity of the molecule and allowed the extraction with organic solvent (Tarcomnicu et al,

2007).

An interesting reaction described by Kline et al (1992) uses 2,3 naphtalene-

dicarboxaldehyde, in presence of cyanide ions as nucleophiles. The substituted

cyanobenzo[f]indole obtained was measured by fluorescence. Alendronate was determined

from urine samples using this technique. The same authors studied also the use of acetyl-

penicillamine as nucleophile for improved sensitivity; electrochemical detection was

preferred over fluorescence.

Introducing a chromophore in order to get sensitive UV or fluorescence measurements is

also possible post-column, after the separation by ion-exchange chromatography. Daley-

Yates et al (1989) described such a derivatization with ammonium persulphate and

molybdenum ascorbate; a phosphomolibdate detected in visible (820 nm) was obtained.

Nevertheless, in the LC methods above presented, although the separation is performed on

octadecyl stationary phase, the mobile phases consisted of phosphate, pyrophosphate,

citrate buffers in combination with various percentages of organic modifiers like methanol

or acetonitrile. These inorganic buffers, as well as mineral acids, are not suitable for LC-MS.

3.4 Overview on mass spectrometric analysis of biphosphonates

When coupling gas chromatography with mass spectrometry, derivatization is needed in

order to obtain volatile compounds. In this case the hydroxyls of the phosphonate group are

silylated or alkylated. Sakiyama et al (2005) described a method for alendronate analysis in

urine that employs double derivatization: first the amino group was reacted directly in the

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biological matrix with isobutyl chloroformate and extracted with organic solvent, then

diazomethane was used to methylate the phosphonate groups. A fused silica capillary

column containing cross-linked OV-1 stationary phase (Quadrex 12mx25mm id, 0.25μm film

thikness) eluted with helium at 1 ml/min was employed for chromatographic separation.

The limit of detection was 20 ng/ml.

The studies conducted for (aminoalkan)phosphonic acids, widely used either as pesticides

or as pharmaceuticals, are good starting points also for bisphoshonate analysis. A large

variety of derivatization approaches is described in the literature.

Silylation with N-methyl N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) was

proposed by Moye and Deyrup (1984) for GC/MS analysis of the herbicide glyphosate and

its metabolite, aminomethylphosphonic acid. In order to obtain high yield of the reaction,

coating of the glassware by exposing it to a diluted solution of phosphoric acid in ethanol

was necessary; however authors reported problems at low ppb levels.

Also in GC/MS pentafluorobenzyl bromide (Palit et al, 2004) and trimethyl orthoacetate

(Kudzin et al, 2003) were used. The two techniques were applied in the determination of

alkyl and aryl bisphosphonic drugs and in trace analysis of organophosphorous pesticides

or nerve agents and their metabolites from biological or environmental samples. Palit et al

(2004) studied the derivatization of methyl phosphonic and pinacolyl methyl phosphonic

acid, degradation products of organophosphorous compounds, with pentafluorobenzyl

bromide (PFBBr) at alkaline pH, in different conditions. They further compared the stability

of the pentafluorobenzyl phosphonic esters with that of the sylylated derivatives, the later

more widely used in GC/MS, but more sensitive to humidity.

The same reaction was tested by our group on two drugs from the bisphosphophonate class,

and the obtained compounds were separated by LC-MS/MS (unpublished data). A phenyl

stationary phase (Supelcosil LC-DP 15cmx3mm, 5μm) was used, eluted in gradient with a

mobile phase containing 50mM ammonium acetate and acetonitrile. High resolution mass

spectrometric detection on a quadrupole-time of flight (QTOF) instrument model Qstar

Pulsar i (AB Sciex), equipped with an electrospray ionisation source (ESI) operated in

negative ions mode was employed. Di- and triesters resulted mainly from the derivatization

of clodronate, as shown in figure 12; further optimisation is needed to increase the yield of

one of the derivatives in order to use this approach for quantitative purposes.

Zhu et al (2006) reported a methodology for the quantification of risedronate and

alendronate by LC-MS/MS in biological samples after derivatization of the phosphonate

groups with diazomethane. As a general approach, the urine or serum samples were loaded

onto Bond-Elut SAX solid-phase extraction (SPE) columns that were washed with water

prior to on-column derivatization with diazomethane. The methylated compounds were

eluted with methanol and separated on an ion-exchange Zorbax 300-SCX column, with

ammonium formate pH 2.5 and acetonitrile (75/25, v/v) as mobile phase. Triple quadrupole

mass-spectrometers model API 4000 or 5000 were used, operated in ESI positive mode. Low

limits of quantification (LLOQs) as of 0.2 ng/ml for risedronate and 1 ng/ml for alendronate

in urine were achieved using this technique.

A general drawback of the methods using diazomethane is the toxic and explosive nature of

the reagent; special care needs to be taken to ensure safety of all operations.

Alternative alkylations of aminoalkyl phosphonic acids could be performed with

fluorinated alcohols/perfluorinated anhydrides mixtures (Deyrup et al, 1985) or trimethyl

orthoacetate (Royer et al, 2000, Kudzin et al (2003).

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Fig. 12. Extracted chromatogram of the negative molecular ions corresponding to the triester

- [MH]- =782.8716 (A) and diester - [MH]- =602.800 (B) obtained in the reaction of clodronate

and PFBBr. Column: Supelcosil LC-DP 15cmx3mm, 5μm, mobile phase: ammonium acetate

and acetonitrile, in gradient. Mass spectrometer: QTOF Qstar Pulsar i, operated in negative

ESI mode.

Deyrup et al (1985) analysed glyphosate (N-phosphonomethylglycine) and its major

metabolite, aminomethylphosphonic acid, from water samples by GC with

flamephotometric, electron capture or mass spectrometric detection, after derivatization

with trifluoroethanol (TFE) and trifluoroacetic anhydride (TFAA). With this approach the

amino and hydroxyl groups are derivatized in the same step. No special glass coating is

needed, as in the case of MTBSTFA, and no particular safety issues are raised, as in the case

of diazomethane derivatization. An LLOQ of 0.5 μg/L in deionized water was obtained

with electron capture detection.

Trimethylorthoacetate (TMOA) was preferred by Royer et al (2000) for the derivatization of

another herbicide, gluphosinate (DL homoalanine-4-yl (methyl)phosphonic acid), and two

of its metabolites, 3-methylphosphonico-propionic and 2-methylphosphonico-acetic acids,

prior to analysis by GC-MS/MS with chemical ionization. This method was applied for the

quantification of gluphosinate and its metabolites in water samples, over a range of 10-150

μg/L.

Kudzin et al (2003) combined anhydride and TMOA approaches for the derivatization of a

series of biologically active aminoalcanphosphonic acids. Briefly, the neat standards or dried

water extracts were dissolved in 0.1 ml of trifluoroacetic acid/trifluoroacetic anhydride

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mixture (1/1, v/v), incubated for 90 min at 40 ºC, then 0.4 ml TMOA were added, followed

by another incubation step at 100 ºC for 120 min. Both amino and hydroxyl groups were

derivatized and the reaction mixtures were analized after preconcentration under vacuum,

by 31P NMR or GC-MS. The derivatization products were stable for several weeks at

ambient temperature and the quantifiable levels by GC-FID (flame ionization detection)

were in the low μmol/L range.

More recently our group studied the application of TMOA derivatization in the analysis of

bisphosphonate drugs. The hydroxyl groups reacted with TMOA in acidic conditions

independently from the presence of amino groups and all tested drugs (alendronate,

pamidronate, etidronate, clodronate, risedronate, ibandronate) were successfully

derivatized. Triethylorthoacetate (TEOA), trimethylorthoformiate (TMOF),

trimethylorthobutirate (TMOB) were also tested as alkylation reagents and found effective

(unpublished data). All reagents do not require special safety measures which is an

important advantage.

Studying these reactions, the expected alkylation products were found, but also other

compounds with higher molecular mass were obtained in the case of bisphosphonates

containing a hydroxyl group at the carbon in the P-C-P bridge. This hydroxyl is also

involved in the reaction probably resulting in cyclic structures. In the case of clodronate, the

carbon in the bridge is substituted with two chlorine atoms and only the tetraalkylated

structure is formed, as it can be seen in Figure 13. When the reagent was TMOF, an increase

of 28 mass units was observed; for TMOA and TEOA the shift was 42, and for TMOB 70 Da.

Based on these results, we have further developed a double derivatization method for the

quantification of alendronate in urine by LC-MS/MS (Tarcomnicu et al, 2007). First, the amino

group was reacted with isobutyl chloroformate (IBCF), to facilitate the extraction of the polar

drug from biological matrix with organic solvent. The dried extract was redissolved in acetic

acid, and incubated with TMOA for 1 h at 100 ºC. The methylation of the phosphonate groups

significantly reduced the polarity of the molecule permitting the separation on a reversed-

phase column (Supelco Discovery HSC18) with a gradient of mobile phase containing formic

acid 0.1% in water/formic acid 0.1% in acetonitrile, which was suitable for LC/MS and

enhanced the ionization in positive mode. The clyclic compounds, as described above, were

obtained with highest yield for alendronate and pamidronate; a product ion spectrum of the

alendronate derivative is presented in Figure 14.

The method vas fully validated according to FDA guidelines over a range of concentrations

of 6.667 – 4860 ng/mL alendronate in urine, and it was applied to a bioequivalence study.

With the help of a switching valve 2 columns were run in parallel, resulting in an analysis

time of 5 min/sample, that was very short compared with HPLC-fluorescence or with GC

methods.

Trimethylsilyl diazomethane (TMSD) was another derivatization reagent successfully used

in LC-MS/MS, in order to analyze ibandronate from plasma samples for pharmacokinetic

applications (Tarcomnicu et al, 2009). A high-throughput method with an LLOQ of 200

pg/mL was optimized and fully validated according to FDA guidelines. Owing to its

structure (N-substituted aminobisphosphonate), ibandronate was isolated from plasma

extracts with the same technique previously proposed for alendronate (Tarcomnicu et al,

2007). To reduce the polarity of the molecule and make it suitable for reversed-phase LC, in

the next step we decided to methylate the phosphonic groups. First, TMOA was tried as

derivatization reagent, but in the case of plasma extracts this was found not as effective as

for urine extracts. TMSD proved to be very reactive also with plasma extracts, as already

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Fig. 13. Clodronate and etidronate derivatization with TMOA; for the latter both hypotheses

(methylation and methylation plus cyclization) are shown.

Fig. 14. Product ion spectrum of the alendronate derivative with IBCF and TMOA ([M+H]+=

448), during chromatographic separation. Column: Supelco Discovery HSC18, 10cmx2.1mm,

5μm, mobile phase: (A) formic acid 0.1% in water and (B) formic acid 0.1% in acetonitrile;

gradient conditions. Mass spectrometer: QTOF Qstar Pulsar i, operated in positive ESI

mode.

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studied by another group (Ranz et al 2008) and compared to diazomethane it is a stable and

safe reagent. The alkylation reaction was carried out for 30 min at 70 ºC and the fully

methylated compound was obtained. The derivatization scheme is presented in Figure 15,

the product ion spectrum of the ibandronate derivative in Figure 16, and Figure 17 shows

selected chromatograms obtained on a blank, calibrator 1 (LLOQ) and calibrator 8 extracted

from plasma. The separation was performed on a Discovery HSC18 column (10cm×2.1mm

i.d., 5μm particle size) and a mobile phase consisting of formic acid 0.1% in water and

formic acid 0.1% in acetonitrile, with a composition gradient. An alternative satisfactory

approach using alkaline mobile phase (ammonium hydroxide 0.05% in water/ammonium

hydroxide 0.05% in acetonitrile) on a Purospher Star RP-18e (3cm×2.1mm, 2.7μm) was also

tested; good peak intensities, similar to the ones with acidic mobile phase, were achieved.

Elution at acidic pH was preferred for this study due to better background, but the basic

mobile phase can be also considered.

Fig. 15. Derivatization scheme of ibandronate and its internal standard, d3-ibandronate.

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Fig. 16. Product ion spectrum of the ibandronate derivative with TMSD - [M+H]+ = 376

obtained with direct infusion via syringe of a 1μg/mL solution water/methanol (1/1, v/v).

Mass spectrometer: quadrupole-linear ion trap API4000QTrap, operated in positive ESI

mode; collision energy: 25V.

Fig. 17. HPLC traces of the MRM transitions selected for ibandronate (left) and d3-

ibandronate (right) recorded on a blank plasma sample (A), plasma spiked with ibandronate

for CAL1—0.2 ng/ml (B) and CAL8—175.0 ng/ml (C) concentrations. Note: The retention

time shown in the figure is relative, as the analyses were performed in overlapping mode;

1.5 min must be added to get the total retention time.

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3.5 Remarks

Considering existing data for the analysis of bisphosphonates, especially with MS detection,

the derivatization approach has given so far the best results. Nevertheless, the new

developments in column manufacturing could open possibilities for the analysis of

underivatized bisphosphonates. A promising direction is represented by HILIC (hydrophilic

interaction liquid chromatography) which is gaining interest in the analysis of polar

compounds, with applications in various fields (A.J. Alpert, 2011, Van Nuijs et al, 2011).

HILIC is a version of normal phase liquid chromatography; the typical mobile phase

consists of acetonitrile with a small percentage of water, while the stationary phases are

polar surfaces (silica, diol, amino, amide, zwitterionic bonded phases). The retention of

polar compounds is not based on liquid-solid partition like in reversed phase separations,

but on a liquid-liquid partition mechanism between the water-deficient bulk eluent and the

water-enriched layer immobilized on the HILIC stationary phases (Alpert et al, 1990).

Therefore polar analytes early eluting in reversed-phase are strongly retained in HILIC

conditions. HILIC is particularly useful in the analysis of aminoglycoside antibiotics (more

details will be provided in the following chapter), other various pharmaceuticals (e.g.

metformin, salicylic and acetyl salicylic acid, tetracyclines), nucleosides, organic phosphates,

peptides and proteins, sugars, warfare agents or drugs of abuse (cocaine, benzoylecgonine)

etc. The use of mobile phases with high percentage of organic solvent in HILIC separation is

beneficial for MS detection, because of enhanced ionization which results in an increased

sensitivity. Methanol, ethanol, 2-propanol, tetrahydrofuran can be used for some

applications. The most common buffers used to control pH and ionic strength are

ammonium acetate or ammonium formate, at low molarities, also favouring the ionization

in MS; only rarely high molarities or non-volatile buffers are required. Another advantage is

the simpler sample preparation, being no need for derivatization. The functional groups on

the phase surface influence the selectivity of the separation, thus the capacity to differentiate

similar compounds. Other interactions which could be involved in HILIC are electrostatic

interaction, hydrogen-bonding, dipole–dipole interaction, molecular shape selectivity, and

even hydrophobic interaction.

The retention mechanisms have been studied by several groups in order to characterize and

classify HILIC stationary phases (Dinh et al, 2011, Kawachi et al, 2011).

4. Analysis of aminoglycoside antibiotics

4.1 Chemical structure and main features

Aminoglycosides are antimicrobial agents used in the treatment of both animals and

humans against aerobic gram-negative bacteria. These molecules are natural products or

semisynthetic derivatives of compounds produced by different varieties of actinomycetes

isolated from soil (Higgins and Kastner, 1967); the class include very important antibiotics

as gentamicin, tobramycin, amikacin, netilmicin, kanamycin, streptomycin and neomycin.

The first drug of the class, Streptomycin, was isolated from a strain of Streptomyces griseus by

Waksman and coworkers in 1944 and it is an important agent for the treatment of tuberculosis.

Gentamicin and netilmicin are derived from species of the actinomycete Micromonospora.

Tobramycin is produced by S.tenebrarius (Chambers, 2006). Amikacin, a derivative of

kanamycin, and netilmicin, a derivative of sisomicin, are semisynthetic products.

The antimicrobial activity of aminoglycosides is based on their ability to selectively inhibit

protein synthesis, in bacteria; the most important pathogens treated with aminoglycosides

are pseudomonas, enterococci, coliforms, and salmonellae.

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The molecule of aminoglycosides presents two or more amino sugars linked by a glycosidic

bridge to a hexose nucleus (aminocyclitol) that is either streptidine (found in streptomycin)

or 2-deoxystreptamine (in all other available aminoglycosides).

The aminoglycosides are divided into the following subclasses (Chambers, 2006):

a. The neomycin family, which includes neomycin B and paromomycin, presents three

amino sugars attached to the central 2-deoxystreptamine.

Neomycin B

b. The kanamycin family (kanamycin A - B, amikacin and tobramycin) presents two amino

sugars attached to the central 2-deoxystreptamine moiety; one of these being a 3-

aminohexose.

Kanamycin A

c. The gentamicin family, including gentamicin, sisomicin and netilmicin, contains two

amino sugars attached to the central 2-deoxystreptamine, one of these being

garosamine.

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Gentamicin C

d. Streptomycin contains streptidine that is not in the central position like in the other

aminoglycosides.

Streptomycin

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4.2 Chromatography of aminoglycosides

Aminoglycoside antibiotics are very polar compounds, highly water soluble, practically

insoluble in hydrophobic organic solvents, lacking chromophores for UV absorption or

fluorescent groups. They have been determined by bioassays, GC–MS and HPLC, in this

case often following derivatization (to improve detection and/or separation), adding ion-

pair agents in the mobile phase or by ion-exchange techniques. More recently, HILIC was

also proposed as worthwhile approach and there is an increasing number of applications; in

literature brief reviews about the application of these methods for the separation of

aminoglycoside antibiotics can be found (Tawa et al, 1998, Isoherranen and Sobak, 1999,

Stead, 2000).

Because of the nonvolatile and very polar nature of aminoglycosides the gas

chromatographic analysis invariably involves derivatization of the amino and hydroxyl

groups of these molecules. trimethylsilylimidazole (TMSI) and heptafluoro-

butyrylimidazole (HFBI) have been used as derivatizing agents for the analysis of

gentamicin, tobramycin, netilmicin, amikacin and paromomycin in serum by Mayhew et al

(1978). Trimethylsilyldiethylamine (TMSDEA) was also tested as derivatization reagent for

both amino and hydroxyl groups but with poor results as repeatability and linearity

(Margosis and Tsuji, 1978).

Owing to their hydrophilic properties, aminoglycosides are not adequately separated also

by reversed-phase liquid chromatography, and therefore derivatization with non polar

agents or mobile phase containing counter-ions reagents, forming ion pairs, have been

widely employed in HPLC.

As derivatization reagents o-phthalaldehyde (OPA), 1-fluoro-2,4-dinitrobenzene (FDNB)

and dansyl chloride are the most commonly used for the analysis of aminoglycosides

(Isoherranen and Sobak, 1999). OPA reacts in the presence of mercaptan or other strong

reducing agents with primary amines to form fluorescent derivatives. The reagent is stable

in different buffer solutions and the reaction has a fast rate at room temperature allowing

the derivatization to be performed either pre- or post- column. FDNB easily reacts with both

primary and secondary amines, in basic conditions, producing UV absorbing derivatives

with maximum absorption wavelength at 365 nm.

The separation columns used for the derivatized aminoglycoside generally contain C8 or

C18 silica as stationary phase; the mobile phase consist of acidic buffers with methanol

and/or acetonitrile.

Gentamicins C1, C1a and C2 were determined as their 2,4-dinitrophenyl derivatives in

plasma and urine using a C18 RP column and as mobile phase 680ml/l acetonitrile-320ml/l

Tris buffer (8.3mmol/l titrated to pH 7.0) (Isoherranen and Sobak, 2000). A HPLC method

for the analysis of tobramycin in urine samples used pre-column derivatization of

tobramycin with fluorescein isothyociante followed by fluorescence detection; the

chromatographic separation was carried out on a Phenomenex Luna C18 column and the

limit of quantitation of the method in urine was 250 ng/mL (Mashat et al, 2008).

Another derivatization agent used for the RP-HPLC analysis of aminoglycoside antibiotics

was phenylisocyanate in the presence of triethylamine. Phenylisocianate groups easily react

with amino groups of aminoglycosides like kanamicin, neomycin and gentamicin (Kim et al,

2001 and 2003); the complex structures were confirmed by ESI-MS.

Clarot et al (2005) described a method for the determination of neomycin sulphate,

framycetin sulphate and other related compounds by evaporative light scattering detection

(ELSD) thus avoiding the need of derivatization for detection. The chromatographic

separation was performed on a Polaris C18 column using a mobile phase with 170 mM

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trifluoroacetic acid; in this case trifluoroacetate ions act like counter-ions forming ion pairs

with aminoglycosides. The ELSD elution of the complexes with antibiotics was confirmed

by MS.

Ion-pair chromatography is probably the most popular approach for aminoglycosides

analysis; when using standard detectors mainly alkyl sulphonates (pentane, heptane and

hexane sulphonates) in acetate or phosphate buffers are selected as counter-ions. For HPLC-

MS applications the volatile fluorinated carboxylic acids are preferred. Pre-column and post

column derivatization with o-phthalaldehyde are often used for the detection of these

molecules also when separated by ion-pair HPLC. As an example, Aubin et al separated

nine aminoglycoside antibiotics (streptomycin, dihydrostreptomycin, neomycin, sisomicin

gentamicin C1, C1a, C2, C2a, netilmicin) on an Atlantis C18 column eluted with 0.02M

potassium dihydrogen phosphate (adjusted to pH 3.0 with phosphoric acid) containing 35

g/l sodium sulfate, 500mg/l sodium octanesulfonate and 15ml/l tetrahydrofuran; the

detection was achieved with a pulsed electrochemical detector. Tobramycin and colistin

sulphate were simultaneous determined from pharmaceutical formulations using a Zorbax

SBC18 column eluted with acetonitrile / water / trifluoroacetic acid and evaporative light

scattering detection (Clarot et al, 2009).

Pulsed electrochemical detection was applied also for the analysis of underivatized

tobramycin (Shruti et al, 2010) and kanamycin by ion-pairing with octansulfonate in

phosphate buffer (Manyanga et al, 2010). A system for the determination of gentamicin by

ion chromatography using pulsed amperometric detector after isocratic elution on a

polystyrene column was developed by Metrohm in 2005.

4.3 Analysis of aminoglycosides by mass spectrometry

The concentrations usually monitored in plasma or serum of patients treated with

aminoglycoside antibiotics are in the low ng/mL range. Due to their non negligible toxicity

in biological systems and to side-effects like inducing resistance in bacteria or alteration of

the normal microbial flora (thus influencing various biological systems), antibiotics are also

carefully monitored in environmental or food and feed samples. Maximum residue limits

(MRLs) have been set in most countries and they are in the low ppb ranges, too. Therefore,

mass spectrometry, being a very sensitive and selective analytical technique is extremely

important in the analysis of these antibiotics. With respect to aminoglycosides, the GC/MS

methods are based on complex and lengthy derivatizations as above mentioned; Preu et al,

1998 adapted the method using trimehtylsilylimidazole and heptafluorobutyrylimidazole

for capillary GC/MS.

LC-MS/MS methods have been preferred recently owing to their high sensitivity and ease

of use compared to GC/MS. Two non-derivatization approaches have been developed in

parallel, reversed phase ion-pair chromatography and HILIC.

Aminoglycoside antibiotics are among the best candidates to be analyzed with HILIC and

such methods were applied for difficult matrices like plasma, kidney or meat. Oertel et al

(2004) described an automated method for the simultaneous quantification of amikacin,

gentamicin, kanamycin, neomycin, paromomycin, and tobramycin in human serum by

HILIC-MS/MS. Separation was carried out on a zwitterionic SeQuant ZIC-HILIC column,

100mm × 2.1mm with a Phenomenex SecurityGuard C18, 4mm × 2mm i.d. The mobile phase

was composed of acetonitrile, 2mM ammonium acetate and formic acid, in gradient

conditions, and data acquisition was performed with a Quattro Micro triple quadrupole

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mass spectrometer equipped with ESI source, operated in positive ions mode. Low limits of

quantification (LLOQ) of 100ng/mL were obtained using 500μL serum extracted by solid

phase extraction (SPE).

Ishii et al (2008) reported a quantification method for seven aminoglycosides from swine

and bovine meat and kidney that also employed a SeQuant ZIC–HILIC column (100 mm ×

2.1 mm, 5 μm) eluted with a mobile phase composed of (A) ammonium acetate 150 mM +

1% formic acid in water and (B) AcN, at a flow rate of 0.3 mL/min. MS detection was

performed in positive mode. LOQs in swine bovine kidney were 25 ng/g for gentamicin, 50

ng/g for spectinomycin, dihydrostreptomycin, kanamycin and apramycin, and 100 ng/g for

streptomycin and neomycin, well below the existing FAO MRLs.

Beside HILIC the most common separation technique for aminoglycoside antibiotics is by

ion-pair chromatography, performed mostly on octyl (C8) or octadecyl (C18) columns,

eluted with mobile phases containing trifluoroacetic (TFA), pentafluoropropionic (PFPA),

heptafluorobutiric (HFBA) or nonafluoropentanoic (NFPA) acids as ion-pair reagents. Good

peak shape and high sensitivities are obtained, thus compensating the negative aspect that

the ion-pair reagents are serious contaminants for the MS interface.

Shen et al (2008) studied the separation of amikacin, streptomycin, spectinomicyn and

gentamicin by ion-pair and HILIC. The ion-pair chromatography was performed on an

Agilent Zorbax SB-C8 (30 X 2.0 mm, 5 μm column), eluted with a mobile phase consisting of

(A) 10 mM NFPA with 10 mM ammonium hydroxide in water and (B) 5 mM NFPA acid

with 5 mM ammonium hydroxide in a 10:90 mixture of water:acetonitrile, with a gradient

from 20% to 90% (B). HILIC was carried out on a SeQuant Zic-HILIC (50 X 2.0 mm, 5 μm)

column, with a mobile phase composed of (A) 5 or 25 mM ammonium formate in water

with formic acid (pH =~2.5) and (B) acetonitrile with 1% (v:v) formic acid; the gradient

started from 90% B and was decreased to 10%B. A simple protein precipitation with cold

acetonitrile was used for clean-up of the different samples (mouse, rat or Guinea pig

plasma) and triple quadrupole (API 4000 or 5000) mass spectrometers were used for

detection. An LLOQ of 20ng/mL for all four antibiotics was achieved with the ion pair

method, while the HILIC method was highly sensitive only for spectinomycin, needing

further optimization for the other analytes.

Amikacin, neomycin and gentamicin in plasma or tissue samples were also analyzed by

Zimmer et al (2008) using a similar approach. They tested HILIC on a Varian Inertsil 5 Si

(50X2.0mm column) with a mobile phase composed of acetonitrile /ammonium acetate 100

mM in water, pH 3, and ion pair separation on a Discovery HS-C18 (2.1x50mm, 3μm)

column eluted with 0.1% HFBA in water / 0.1% HFBA in acetonitrile (all methods were

carried out using a composition gradient). LLOQs of 5 ng/mL were obtained using ion-pair

LC, which also in this case has proven to be more sensitive than HILIC.

Granja et al (2009) described a method for the determination of streptomycin residues in

honey that employs ion-pairing with HFBA in water/acetonitrile (85:15, v/v) as a mobile

phase (isocratic conditions), on a C18 column (Gemini 5 micron C18, 50x2mm). Also

Hammel et al (2009) presented a multi-screening approach for 42 antibiotic residues in

honey and 3 aminoglycosides where among them. HPLC analyses were run on a Zorbax SB-

C18 reverse phase column (2.1×50 mm, 1.8μm). The mobile phases were constituted with

solvent A:water containing 1mM NFPA mixed with 0.5% formic acid (v/v) and solvent B:

acetonitrile/methanol (50/50, v/v) containing 0.5% formic acid (v/v).

The following paragraphs present the practical experiments of our group on aminoglycoside

antibiotics by LC-MS/MS (unpublished data). The first method was developed for residues

of streptomycin in honey, by ion-exchange chromatography, on a Grom-Sil 300WCX

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(100x4.6mm, 7μm) eluted in gradient with (A) methanol/water (60/40, v/v) and (B)

ammonium acetate 500mM in methanol/water (60/40, v/v); the initial mobile phase was

100% A. The honey sample was simply diluted 1/1 with water and injected; detection was

achieved with an API 4000 triple quadrupole mass-spectrometer operated in positive ESI

ionization. The calibration curve in honey was built over a range of 10 - 250 ng/mL and

gentamicin was used as internal standard. In order to improve the sensitivity and

robustness of the determination, later we switched to ion-pair chromatography on a

pentafluorophenylpropyl column (Discovery HSF5 100x2.1 mm, 5μm), using a gradient of

PFPA 0.2% in water/PFPA 0.2% in acetonitrile as mobile phase. An LLOQ of 3.3 ng/mL was

obtained with the same instrument set-up and same sample preparation; gentamicin was

replaced as internal standard by cimetidine. Figure 18 shows two examples of

chromatograms, recorded on honey samples.

Fig. 18. HPLC traces of the MRM transitions selected for streptomycin and its internal

standard cimetidine, recorded on a spiked honey sample – concentration 90 ng/mL (top),

and on an unknown sample (bottom chromatograms) that was obviously contaminated with

streptomycin. Column: Discovery HSF5 (10cmx2.1mm, 5μm), mobile phase: A) 0.2% PFPA

in water and B) 0.2% PFPA in acetonitrile; flow: 0.3 ml/min; injection volume: 50μL. Mass

spectrometer: Triple quadrupole API4000, operated in ESI positive mode.

The same method was later adapted for the analysis of neomycin in ophthalmic solutions,

and streptomycin was used as internal standard. The samples were just diluted 1:1000 with

the initial mobile phase (water/acetonitrile (95/5, v/v) + 0.2 % pentafluoropropionic acid)

and injected in the LC-MS system; being the concentrations quite high (1-5 μg/mL) no

further optimization was needed. An example of chromatogram is presented in Figure 19.

Another approach in aminoglycoside analysis by HPLC remains pre-column

derivatization, aiming to reduce polarity and make the compounds suitable for reversed-

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phase separations. Based on a previous research of Lauser and Bergner-Lang (1995), we

tested in our laboratory the derivatization of neomycin with FMOC. The reaction needs to

be optimized, because partial and fully substituted derivatives (all six amino groups

reacted with FMOC) are formed and in order to obtain a better sensitivity the equilibrium

should be ideally directed towards one product. We performed the chromatographic

separation on a C8 column eluted with water, acetonitrile and formic acid and

preliminary tests on plasma extracts have given promising results. The chromatographic

traces corresponding to the fully substituted neomycin and paromomycin derivatives (the

selected internal standard) on plasma samples are shown in Figure 20, while the product

ion spectrum of neomycin derivative in Figure 21.

Fig. 19. Chromatographic traces of neomycin and its internal standard streptomycin,

recorded on spiked sample (concentration 1 μg/ml). Column: Discovery HSF5

(10cmx2.1mm, 5μm), mobile phase: A) 0.2% PFPA in water and B) 0.2% PFPA in acetonitrile;

flow: 0.3 ml/min; injection volume: 10μL. Mass spectrometer: quadrupole-linear ion trap

API4000 QTrap, operated in ESI positive mode.

Last by not least, we have studied HILIC for the determination of vancomycin in plasma,

using neomycin as internal standard. The column employed was an Ascentis Express HILIC

(100x2.1mm, 2.7μm), the separation being carried out in isocratic conditions with

acetonitrile/water (90/10, v/v) containing 0.1% formic acid. A good sensitivity in the range

of few ng/mL, adequate for therapeutic drug monitoring, was obtained.

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Fig. 20. The MRM transitions of neomycin and paromomycin (internal standard) derivatives

with FMOC, obtained after injection of a derivatized plasma extract (spiked concentration

1ng/mL). Column: Ascentis Express C8 (100x2.1mm, 2.7 μm); mobile phase: A)

water/acetonitrile (90/10, v/v) and B) acetonitrile, with composition gradient; injection

volume: 50μL. Mass spectrometer: triple quadrupole API 4000 operated in ESI positive

mode.

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522

Fig. 21. Product ion spectrum of the neomycin derivative with FMOC - [M+H]+ = 1948

obtained with direct infusion via syringe of a 1μg/mL solution in water/methanol (1/1,

v/v). Mass spectrometer: triple quadrupole API4000, operated in positive ESI mode;

collision energy: 30V.

5. Conclusions

The data presented above clearly show that, despite the fact that highly polar compounds

present optimal characteristics to be analyzed by MS, they are difficult molecules due to

critical separation problems generated by the need to employ mobile phases compatible

with the ionization source and process. It is therefore essential, when developing a new

method for such compounds, to first consider the available options to get an optimal

separation compatible with the MS interfacing.

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Between the various options HILIC is gaining a lot of interest as a separation for highly

polar compounds; the development of several columns dedicated to this technique are

widening a lot its application and the growing mass of publications in the field is a prove.

HILIC employs high percentage of organic solvent in the mobile phase usually leading to

enhanced ionization and lesser contamination of the ion source.

Ion exchange remains an interesting alternative especially when considering weak

exchanger stationary phases; in comparison to HILIC however the technical development is

moving much slower. Mixed mode columns (ion exchange + reversed phase) are an

interesting opportunity to get improved separation methods.

The use of ion-pair reagents in connection with reversed-phase columns is another

opportunity but it presents a few pitfalls: restrictions on ionization polarity (in general ion-

pair suppress ionization in the same polarity, i.e. quaternary ammonium salt and positive

ionization), often complex spectra, reduced analytical sensitivity and finally a tendency to

contaminate the ionization source and quadrupoles. It is however interesting, see the GAGs

examples, that ion-pair reagents can, in some cases, positively help the ionization reducing

source fragmentation.

Globally the previously indicated strategies are preferable whenever possible. As a final

option derivatization has to be considered, without meaning that it is the last desirable

approach. It has to be considered especially in case of amphoteric compounds bearing both

positive and negative charges; such compounds are generally difficult to extract and the

ionization is not optimal; aminobisphosphonates are a good example in this context. The

development of derivatization methods can be time consuming but often very solid

methods and a quite good analytical sensitivity can be obtained. It is interesting to see a

continuous development for obtaining optimal derivatization reagents for LC-MS detection.

GC derivatization agents are indeed aiming to get non-polar and volatile compounds,

aspects not always desirable in LC-MS, while fluorescence and UV detection reagents can be

chemically very complex without favoring the ionization process.

The analytical approach described can be usefully applied as well to other classes of highly

polar compounds (for examples polynucleotides, phosphorylated carbohydrates, several

pharmaceuticals).

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www.intechopen.com

Tandem Mass Spectrometry - Applications and Principles

Edited by Dr Jeevan Prasain

ISBN 978-953-51-0141-3

Hard cover, 794 pages

Publisher InTech

Published online 29, February, 2012

Published in print edition February, 2012

InTech Europe

University Campus STeP Ri

Slavka Krautzeka 83/A

51000 Rijeka, Croatia

Phone: +385 (51) 770 447

Fax: +385 (51) 686 166

www.intechopen.com

InTech China

Unit 405, Office Block, Hotel Equatorial Shanghai

No.65, Yan An Road (West), Shanghai, 200040, China

Phone: +86-21-62489820

Fax: +86-21-62489821

Tandem Mass Spectrometry - Applications and Principles presents comprehensive coverage of theory,

instrumentation and major applications of tandem mass spectrometry. The areas covered range from the

analysis of drug metabolites, proteins and complex lipids to clinical diagnosis. This book serves multiple groups

of audiences; professional (academic and industry), graduate students and general readers interested in the

use of modern mass spectrometry in solving critical questions of chemical and biological sciences.

How to reference

In order to correctly reference this scholarly work, feel free to copy and paste the following:

Luigi Silvestro, Isabela Tarcomnicu and Simona Rizea Savu (2012). HPLC-MS/MS of Highly Polar Compounds,

Tandem Mass Spectrometry - Applications and Principles, Dr Jeevan Prasain (Ed.), ISBN: 978-953-51-0141-3,

InTech, Available from: http://www.intechopen.com/books/tandem-mass-spectrometry-applications-and-

principles/hplc-ms-ms-of-highly-polar-compounds

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